Methods for processing biological samples by means of laser microdissection have been in existence since the mid-1970s (see e.g. Isenberg, G. et al.: Cell surgery by laser micro-dissection: a preparative method. Journal of Microscopy, Vol. 107, 1976, pages 19-24) and have been continuously developed ever since.
In laser microdissection, cells, tissue regions and the like can be isolated from a sample (“object”, “preparation”) and obtained as dissectates. A particular advantage of laser microdissection is that the sample comes into brief contact with the laser beam, and this barely alters the sample. In this context, the dissectates can actually be obtained in various ways (see e.g. Bancroft, J. D. and Gamble, M.: Theory and Practice of Histological Techniques. Elsevier Science, 2008, page 575, Chapter “Laser Microdissection”).
In known methods, for example, a dissectate can be isolated from a sample by means of an infrared or ultraviolet laser beam, which dissectate falls into a suitable dissectate collection container as a result of gravity. In the process, the dissectate can also be cut out from the sample together with an adherent membrane. By contrast, in laser capture microdissection, a thermoplastic membrane is heated by means of an appropriate laser beam. In the process, the membrane fuses with the desired region of the sample and can be removed in a subsequent step by being torn off. A further alternative is to fix the dissectate to a lid of a dissectate collection container by means of the laser beam. In known inverted microscope systems for laser microdissection, dissectates catapulted upwards can also be fixed to the base of a dissectate collection container provided with an adhesive coating.
Known microscope systems for laser microdissection, as known for example from WO 98/14816 A1, comprise an incident-light device, into the beam path of which a laser beam is coupled. The laser beam is focused onto the sample, which rests on a microscope stage that can move automatically by means of a motor, by the microscope objective used in each case. A cut line is produced by the microscope stage being moved during the cutting in order to move the sample relative to the stationary laser beam. This, however, is disadvantageous inter alia in that the sample cannot be easily viewed while the cut line is being produced since said line moves in the field of vision and the image appears blurred.
Laser microdissection systems comprising laser deflection devices or laser scanning devices designed to deflect the laser beam or the incident point thereof over the stationary sample are therefore more advantageous. Laser microdissection systems of this type, which also offer particular advantages in the context of the present invention, are explained in detail below. A particularly advantageous laser microdissection system which comprises, in the laser beam path, a laser scanning device having glass wedges which can move relative to one another, is described for example in the above-mentioned patent EP 1 276 586 B1.
In both cases, that is both in laser microdissection systems in which the microscope stage is moved and in laser microdissection systems which comprise a laser scanning device, pulsed lasers are generally used, a hole being made in the sample by each laser pulse. A cut line is produced by making such holes next to each other, optionally with an appropriate overlap.
The laser microdissection can be used to obtain single cells or defined tissue regions, in other words dissectates of nucleic-acid-containing samples. Corresponding dissectates can then undergo various molecular biology analysis methods.
To check for DNA damage (in particular individual or double strand breakages, multiplications, deletions, dimerisations, etc.) in single cells or particular tissue regions, single-cell gel electrophoresis is known, for example (see e.g. Wood, D. K. et al.: Single-cell trapping and DNA damage analysis using microwell arrays, Proc. Natl. Acad. Sci. USA, Vol. 107, 2010, pages 10.008-10.013). Single-cell gel electrophoresis (also referred to as the comet assay) is based on the fact that corresponding DNA damage, for example strand breakages in the DNA, cause changes to the geometric properties thereof and thus the migration behaviour or mobility in the electrophoresis.
In this respect, the general observations are that damaged DNA fragments are more mobile in the conventionally used agarose gels than undamaged DNA fragments. This can be seen in a comet-tail-like migration image. By means of single-cell gel electrophoresis, a plurality of different DNA lesions can be detected, it also being possible, for example, to use DNA repair enzymes or other reagents to show up damage which is undetectable per se. An overview of this can be found in the above-mentioned article by Wood et al.
However, single-cell gel electrophoresis can conventionally only be carried out at low throughputs and with relatively poor reproducibility. The image processing and analysis methods used are complex, laborious and potentially susceptible to errors. The article by Wood et al. proposes a method for high-throughput single-cell gel electrophoresis, wherein an electrophoresis gel having a number of gel pockets is used and can subsequently undergo standard high-throughput screening techniques. In particular, however, it is not possible to use the method disclosed therein to specifically place particular cells or cell types into specific gel pockets. In the method, the entire gel is covered with a cell suspension and the respective cells settle at random in the gel pockets provided in each case.